Int J Pharm Pharm Sci, Vol 6, Issue 8, 263-268Original Article

IN VITRO ANTIOXIDANT AND ANTICANCER ACTIVITY OF CARDIOSPERMUM HALICACABUM L. AGAINST EAC CELL LINE

AISHWARYA V., #SHEIK ABDULLA S., DHEEBA B, RENUKA R

Department of Chemistry and Biosciences, SASTRA University, Kumbakonam campus, Tamil Nadu, South India
Email: sheikhaneef@src.sastra.edu

Received: 17 May 2014 Revised and Accepted: 21 Jun 2014

ABSTRACT

Objective: To investigate the in vitro antioxidant and anticancer activity of chloroform and ethanol extracts of Cardiospermum halicacabum L. leaves.

Methods: Phytochemicals were analysed by using standard methods. In vitro antioxidant studies were carried out for the chloroform and ethanol extracts of the Cardiospermum halicacabum using various free radical models such a DPPH, Reducing power assay, nitric oxide scavenging, hydrogen peroxide (H2O2) scavenging, super oxide scavenging activity and ABTS. In vitro cytotoxic assay such as trypan blue dye exclusion and MTT assays were carried out against EAC cell line.

Results: Preliminary phytochemical analysis of the Cardiospermum halicacabum L. was carried out and it revealed the presence of alkaloids, coumarine, flavones, saponins, steroids, sugar, tannins and terpenoids. The results revealed that the chloroform extract has significant antioxidant potential than ethanol extract. The result revealed that the chloroform extracts of Cardiospermum halicacabum L. showed pronounced anticancer activity against Ehrlich Ascites Carcinoma (EAC) cell line than ethanol extract.

Conclusion: The result of the present study concluded that the chloroform extract of Cardiospermum halicacabum L have significant antioxidant and anticancer activity then the ethanolic extract. The potential antioxidant and anticancer activity of Cardiospermum halicacabum L might be due to the presence of phytochemicals.

Keywords: Cardiospermum halicacabum L, Ehrlich Ascites Carcinoma, Free radicals, Antioxidant.

INTRODUCTION

Cancer is a growing public problem whose estimated worldwide new incidence is about 6 million cases per year. It is the second major cause of deaths after cardiovascular diseases. Cancer is a general term applied of series of malignant diseases that may affect different parts of body. These diseases are characterized by a rapid and uncontrolled formation of abnormal cells, which may mass together to form a growth or tumor, or proliferate throughout the body, initiating abnormal growth at other sites. If the process is not arrested, it may progress until it causes the death of the organism. These cells are born due to imbalance in the body and by correcting this imbalance the cancer may be treated [1, 2]. Many things are known to increase the risk of cancer including tobacco use, dietary factors, certain infections, exposure to radiation, lack of physical activity, obesity and environmental pollutants. These factors can directly damage genes or combine with existing genetic faults within cells to cause cancerous mutations. Approximately 5–10% of cancers can be traced directly due to inherited genetic defects [3]. Chemicals such as tobacco smoke contain over fifty known carcinogens, including nitrosamines and polycyclic aromatic hydrocarbons. Alcohol is also a carcinogen [4]. Excessive free radicals produced during cellular metabolism can attack the deoxyribose DNA backbone and bases, which leads to cytotoxicity or mutation and finally it causes cancer. Free radicals also a factor which causing all types of cancer [5].

There is a necessity for research to search of new compounds with cytotoxic activity, as the treatment of cancer. The available anticancer drug is often unsatisfactory due to the problem, which cause cytotoxicity to the normal cells along with cancer cells. Plants are considered as the valuable sources of bioactive compounds with antioxidant activity, which produce certain substances that have effects on living animal cells. Some extracts rich in apigenin (found in parsley, bell pepper, etc.) causes cancer cells to undergo apoptosis (programmed cell death) in in vitro studies [6]. The aim of the present study is to evaluate the in vitro antioxidant and antitumor activity of Cardiospermum halicacabum L.

In recent years, there has been considerable emphasis on the identification of plant products with antioxidant property, as free radicals are considered to play a major role in most of the diseases including cancer. The medicinal value of the chosen plant Cardiospermum halicacabum L bark has been extensively worked out. However, its therapeutic efficacy in antioxidant and antitumor activity has not been evaluated. Cardiospermum halicacabum L. is the member of the family Sapindaceae. The major chemical constituents of C. halicacabum reported to contains (+)-pinitol, β-sitosterol, β-sitosterol-β-D-galactoside, apigenin-7-O-glucuronide, arachidic acid, chrysoeriol-7-O-glucuronide, linoleic acid, lutrolin-7-O-glucudonide, stearic acid [7]. Cardiospermumhalicacabum L. was used to treat various diseases such as skin diseases (rashes, itching, skin irritation, etc.), dandruff, rheumatoid arthritis, gastrointestinal diseases, respiratory tract diseases, urogenital diseases, etc.[8].

MATERIALS AND METHODS

Plant Collection

Plant source selected for the present study was Cardiospermum halicacabum L. The leaves of the selected plant were collected from in and around Thanjavur. The collected leaves were washed with tap water and shade dried under room temperature and grounded to fine powder using blender. The powder was preserved in an air tight bottle for further studies.

Preparation of plant extracts

Preparation of Chloroform Extract

250g of coarse powder of Cardiospermum halicacabum L. was soaked in chloroform for 48 hours. Filtered the solution and the filtrate was evaporated to the dryness. The residue was dissolved in chloroform and used for study [9].

Preparation of Ethanol Extract

250g of dried plant material was soaked in ethanol for 48 hours. Filtered the solution and the filtrate was evaporated to the dryness. The residue was dissolved in ethanol and used for study [10]. Preliminary phytochemical screening of drug powder and various extracts were carried out as per the standard textual procedure [11].

In vitro Antioxidant activity

Antioxidant activity measured by using DPPH radical scavenging assay method [12], Reducing Power assay [13], Hydrogen peroxide radical scavenging activity [14], Nitric oxide scavenging activity[15], Superoxide radical scavenging activity [16] and ABTS Radical scavenging activity [17]. Tests were carried out in triplicate for 3–5 separate experiments. The amount of extract needed to inhibit free radicals concentration by 50%, IC50, was graphically estimated using a nonlinear regression algorithm.

Anticancer screening

Experimental animals [18]

Healthy adult Swiss Albino mice of both sexes, weighing 25-35g were obtained from Tamil Nadu Veterinary and Animal Sciences University, Chennai. The animals were allowed to acclimatize under laboratory conditions for a period of 5 days prior to the experiment. Animals were housed in standard polypropylene cages. Six animals were housed per cage, so as to provide them with sufficient space and to avoid unnecessary morbidity and mortality. Animals were maintained under standard condition of 12:12- hour’s light/ dark cycle and at an ambient temperature at 23 ± 2°C, with 65 ± 5 % humidity. Animals were fed with standard rat chow pellet obtained from Sai Durga Foods and Feeds, Bangalore, India. All studies were conducted according to the ethical guidelines of CPCSEA after obtaining necessary clearance from the committee (Approval No: 790/03/ac/CPCSEA).

Maintenance of EAC Cells [19]

Ehrlich Ascites Carcinoma cell line was obtained from Amla Cancer Research Centre, Thrissur and was maintained by weekly intra-peritoneal inoculation of 1X106cells/mouse.

In Vitro Cytotoxicity assays

Trypan Blue [20]

Short- term in-vitro cytotoxicity was assessed using Ehrlich Ascites Carcinoma cell line by incubating different concentrations of chloroform and ethanol extracts of Cardiospermum halicacabum L. at 37°C for 3 hours. The tumor cells were aspirated from the peritoneal cavity of tumor bearing mice using an insulin syringe and transferred to a test tube containing isotonic saline. The cells were then washed in normal saline and the cell number was determined using a haemocytometer and adjusted to1x106 cells/ml. For the cytotoxity assay, different concentrations of the extract (50-1000 µg/ml) were added to each tube and the final volume was adjusted to 1ml with saline. Control tubes were maintained with the saline and tumor cells, but without the plant extract. All the tubes were incubated at 370C for 3 hours. After incubation 0.1ml of 0.2% tryphan blue dye in isotonic saline was added to a watch glass along with 0.1ml of test sample and the number of viable (unstained) and non-viable (stained) cells were counted using haemocytometer.

MTT Assay [21]

Increasing concentrations of chloroform and ethanol extracts of Cardiospermum halicacabum L. were added to the cells and incubated at 37ºC for 24 hrs in CO2 incubator with 5% CO2. The media was replaced with a fresh growth medium along with 20 µl of 3(-4, 5-dimethyl thiazol-2-yl) 2, 5 diphenyl tetrazolium bromide (MTT, sigma). Again it was incubated for 4 hrs at 37ºC. After incubation purple precipitate was clearly visible under the inverted microscope then the growth medium was removed and 200µl of 0.1% 0.1N acidic isopropyl alcohol was added to the cells to dissolve the formazon crystals. Then the covered plates were kept in the dark at 18-24ºC per overnight. The samples were then drawn every 2 hours and observed the reading at 570nm. If the reading was low the plates were returned for incubation. Each experiment was conducted in triplicate. The average was calculated and compared with the control test samples. The percentage growth inhibition was calculated using the following formula.

RESULTS AND DISCUSSION

Cancer results from a series of molecular events that fundamentally alter the normal properties of cells. In cancer cells the normal control systems that prevent cell overgrowth and the invasion of other tissues are disabled [22].Current cancer chemotherapy can damage or kill the rapid dividing healthy cells and causes serious side effects such as neutropesia, anemia, etc. In addition, the cost of chemotherapy drug is high. Natural compounds may reduce these problems. Currently, a few plant products are being used to treat cancer effectively [23].

In the present study attempt was made to develop a novel anticancer drug from a common plant source. From the literature, we found that plant belonging to the family Sapindaceae is well documented for their anticancer potential[24].Hence in the present work a commonly available plant belonging to the family Sapindaceaea nd botanically equated as Cardiospermumhalicacabum L. was selected. Cardiospermumhalicacabum L. was well known for its medicinal values. The preliminary phytochemical screening of various extracts of the Cardiospermumhalicacabum L. was analyzed. Chloroform extract contains alkaloids, coumarin, flavones, quinones, saponins, steroids and tannins. Ethanol extract showed the presence of alkaloids, coumarin, flavones, quinones, saponin, terpenoids, steroids and sugar.

The chloroform and ethanol extracts of the leaves of selected plant material was screened for its antioxidant potential using various free radical models such a DPPH, Reducing power assay, NO, H2O2, superoxide radical sacvenging and ABTS and anticancer activity against EAC cell line.

DPPH is a relatively stable free radical and the assay determines the stability of chloroform and ethanol extracts of Cardiospermumhalicacabum L.to reduce DPPH radical to the corresponding hydrazine by converting the unpaired electrons to paired ones. Antioxidants can act by converting the unpaired electrons to paired ones. Chloroform and ethanol extracts of plant was allowed to react with stable DPPH radicals is determined by decrease in its absorbance at 517nm, which is induced by an antioxidant present in the extracts after 10 minutes. The concentration of antioxidant needed to decrease the initial DPPH concentration by 50% is known as IC50 value. The IC50 value of the extracts was found to be 214µg/ml for chloroform and 295µg/ml for ethanol extract. The scavenging of DPPH radical by the extracts was compared with standard ascorbic acid (Table 1).

DPPH radical scavenging activity of methanol extract of leaves of the plant Adiantum philippense L. was found to increase with increasing concentration of the extract. This assay was based on the ability of1,1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, to decolorize in the presence of antioxidants, which denotes the plant has antioxidant potential [25].

Thymelaea hirstuta has been shown that the scavenging effect of DPPH sharply increased with increase in concentration and the results are compared with standard ascorbic acid. The antioxidant potential of T. hirstuta was determined by the effective scavenging of DPPH by the plant extracts [26]. DPPH has been used to evaluate the antioxidative activity of natural products in organic solvents. Hydrogen-donating abilityof the antioxidant is responsible for its free radical-scavenging activity. The DPPH method is based on the reduction of DPPH solution to DPPH-H in the presence of a hydrogen-donating antioxidant. The aqueous extract of Ricinus communis L. leaves and the isolated compounds showed a concentration dependent antiradical activity. The aqueous extract of the leaves was found to possess radical-scavenging activity. Ascorbic acid was used as positive control [27].

Fe (III) reduction is often used as an indicator of electron donating activity, which is an important mechanism of antioxidant activity. In the reducing power assay, the presence of antioxidant in the test drug converts the Fe3+ to Fe2+ by donating electron. Amount of Fe2+ complex formed can be measured at 700nm. Increasing absorbance indicated an increase in reductive ability. Table 2 shows the reducing power of the extracts also increased with increase in their concentrations. The IC50 values for the chloroform and ethanol extracts are 325µg/ml and 400µg/ml respectively. Reducing power is one of the mechanisms for the possible antioxidant activity and may serve as significant indicator of potential antioxidants. Ethyl acetate fraction of Hybanthus enneaspermus showed good concentration dependent manner with a perfect reducing power, thus indicating a good electron donors and ability to terminate radical scavenging reaction [28]. The methanol extracts of seeds of the Citrullus colocynthis has a significant reducing power, which indicates the antioxidant potential of the plant. It reduces the Fe3+ to Fe2+ by donating an electron. The antioxidant potential increases with increase in the concentration of the extract. The results are compared with standard ascorbic acid [29].

Hydrogen peroxide scavenging activities of the extracts are given in Table 9 and are compared with the standard ascorbic acid. The chloroform and ethanolic extracts of Cardiospermum halicacabum shows their activities at different concentration. H2 O 2 is highly important because of its ability to penetrate biological membranes. H2 O2 is not very reactive and it is a weak oxidizing agent, but it can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cells. It can cross cell membranes rapidly, once inside the cell, H2O2 can probably react with Fe2+ and possibly Cu2+ ions to form hydroxyl radical and this may be the origin of many of its toxic effects. It can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups [30].

Table 1: DPPH radical scavenging assay of chloroform and ethanolic extracts of Cardiospermum halicacabum L. leaf

S. No. Concentrations (μg/ml) DPPH radical Scavenging activity (%)
Standard (Ascorbic acid) Chloroform extract Ethanol extract
1. 50 25.22±1.76 10.70±0.74 20.00±1.4
2. 100 30.14±2.10 25.20±1.76 28.50±1.99
3. 150 36.22±2.53 35.00±2.45 35.48±2.48
4. 200 44.06±3.08 42.80±2.99 43.24±3.02
5. 250 55.40±3.87 53.50±3.74 54.20±3.79

IC50 value of chloroform extract = 214 µg/ml, IC50 value of ethanol extract = 295 µg/ml


Table 2: Reducing power assay of chloroform and ethanolic extracts of Cardiospermum halicacabum L. leaf

S. No. Concentrations (μg/ml) Reducing power (%)
Standard (Ascorbic acid) Chloroform extract Ethanol extract
1. 100 22.04±1.54 20.00±1.4 20.00±1.4
2. 250 45.26±3.16 44.50±3.11 42.80±2.99
3. 500 65.98±4.61 62.60±4.38 55.50±3.88
4. 750 78.10±5.46 75.00±5.25 60.11±4.20
5. 1000 88.96±6.22 84.00±5.88 66.65±4.66

IC50 value of chloroform extract = 325 µg/ml, IC50 value of ethanol extract = 400 µg/ml

At minimal concentration (200µg/ml) of the chloroform and ethanol extracts showed 18.18% and 16.66% H202 scavenging activity, respectively. At maximal concentration the chloroform and ethanol extracts showed potent H202 scavenging activity 77.27% and 70.00% respectively. The IC50 values for the chloroform and ethanol extracts for scavenging of H2O2 were 510µg/ml and 580µg/ml (Table 3).

Leaf extract of Lantana camara showed good hydroxyl radical scavenging activities (45%-73%)at a concentration of 0.2-0.8 mg/ml in the reaction mixture. Leaf extract showing hydroxyl radical-scavenging activity was increased with increasing concentration of the extract sample. Leaf fraction of L. camara had higher activity but lower than that of ascorbic acid [31]. Hydrogen peroxide itself is not very harmful, but sometimes it may harmful to the cell due to the fact that it may give rise to hydroxyl radical present in the cells. Therefore, eradication of H202is the superior step for antioxidant defense in cell or food system. The various extracts of the Hybanthus enneaspermus(Linn.) plant have significant antioxidant potential and it scavenge H202 potentially [28]. In the present study, the extracts compete with oxygen to react with nitric acid and thus inhibit the generation of the anions. In the experiment, the NO was generated from sodium nitro prusside in aqueous solution at physiological pH was incubated with different concentration of plant extracts (100 - 1000µg/ml).

NO produced from sodium nitro prusside react with O2 to formed nitrite which reacts with griess reagent forms chromophore which was read at 546 nm [32].

The decrease in the OD of test drug incubation directly correlated with inhibition nitric oxide production. From Table 4, the percentage of inhibition of nitrite by the extract of the plant drug was found to be dose dependent one, the IC50 values for the chloroform and ethanolic extracts was 475µg/ml and 450µg/ml respectively. The results were compared with standard ascorbic acid. This may be due to the presence of antioxidant principle present in the extracts, which inhibits the binding of oxygen with nitric oxide. Nitric oxide (NO) is an important chemical mediator generated by endothelial cells, macrophages, neurons, etc. and involved in the regulation of various physiological processes. Excess concentration of NO is associated with several diseases. Oxygen reacts with the excess nitric oxide to generate nitrite and peroxy nitrite anions, which act as free radicals[33]. The aqueous extract of Lantana camara leaves and the isolated compounds showed a concentration dependent antiradical activity. The aqueous extract of the leaves was found to possess NO radical-scavenging activity. The antioxidant activity of L. camarawas increased with increase in plant extract concentration. L. camaraact in a dose dependent manner. Ascorbic acid was used as positivecontrol[31].

Table 3: H2O2 radical scavenging assay chloroform and ethanolic extracts of Cardiospermum halicacabum L. leaf

S. No. Concentrations (μg/ml) H2O2 radical Scavenging activity (%)
Standard (Ascorbic acid) Chloroform extract Ethanol extract
1. 200 20.38±1.42 18.18±1.27 16.66±1.16
2. 400 39.32±2.75 36.36±2.54 33.33±2.33
3. 600 63.34±4.43 59.09±4.13 53.35±3.73
4. 800 75.30±5.27 72.72±5.09 63.31±4.43
5. 1000 78.42±5.48 77.27±5.40 70.00±4.90

IC50 value of chloroform extract = 510 µg/ml, IC50 value of ethanol extract = 580 µg/ml


Table 4: Nitric oxide scavenging assay chloroform and ethanolic extracts of Cardiospermum halicacabum L. leaf

S. No. Concentrations (μg/ml) Inhibition of NO production (%)
Standard (Ascorbic acid) Chloroform extract Ethanol extract
1. 100 38.32±2.68 36.33±2.54 15.70±1.09
2. 250 43.00±3.01 40.00±2.8 26.30±1.84
3. 500 55.16±3.86 53.32±3.73 52.68±3.68
4. 750 70.12±4.90 66.61±4.66 68.41±4.78
5. 1000 86.86±6.08 80.02±5.60 84.22±5.89

IC50 value of chloroform extract = 450 µg/ml, IC50 value of ethanol extract = 475 µg/ml

Superoxide radical is known to be very harmful tocellular components as a precursor of more reactive oxidative species, such as singlet oxygen and hydroxyl radicals. Furthermore, superoxide radical is considered to play an important role in the peroxidation of lipids [28]. Therefore, studying the scavenging effects of Cardiospermum halicacabum L. on superoxide radicals is one of the most important waysof clarifying the mechanism of antioxidant activity. The inhibition of O2- was found to be concentration dependentand the percentage of inhibition was increased with the increased in concentration. The IC50value of the chloroform and ethanolic extracts was found to be 380μg/ml and 460μg/ml, respectively (Table 5). These results indicated that the Cardiospermum halicacabum L. had a notable effect in scavenging superoxide radicals. Inhibition of superoxide generation by Cardiospermum halicacabum L. may be due to the presence of phytochemicals such as flavonoids, alkaloids and phenol.

Superoxide anion plays an important role in plant tissues and it involved in the formation of other cell-damaging free radicals. The methanol extract of Dimocar puslonganLour.has potent antioxidant capability was detected by the scavenging potential of the superoxide anion. The plant has high phenolics. It is known that the hydroxyl group of the phenolics contributes to superoxide anion scavenging activity by their electron donation [22]. The chloroform extract of the Ricinus communis L. has a potent superoxide anion scavenging ability which confirmed that the plant has antioxidant property. Naturally medicinal plants have the antioxidant potential. The superoxide anion scavenging potential of the plant increased with increase in the plant extract concentration. The antioxidant potential of the Ricinus communis L. to scavenge superoxide anion may due to the presence of alkaloids and flavonoids[27].

The ABTS assay is based on the inhibition of the absorbance of radical cation (ABTS +), which has a characteristic wavelength at 734nm by antioxidants. The principle behind the technique involves the reaction between ABTS and potassium per sulphate to produce the ABTS radical cation (ABTS+) which is a blue green chromogen. In the presence of antioxidant reductant, the coloured radical is converted back to coloureless ABTS. The order of ABTS radical scavenging activity of the extract was almost similar to that observed for DPPH. The IC50value of the chloroform and ethanol extracts was found to be 530μg/ml and 570μg/ml respectively (Table 6). The antioxidant potential of the aqueous extract of the Thymelaea hirsuta L. was determined by using the ABTS assay and it has showed a potent antioxidant property. The ABTS radical was significantly scavenged by the plant extract. ABTS react with potassium per sulphate to produce ABTS+,which was scavenged by the plant extract [26].

Table 5: Superoxide radical scavenging assay of chloroform and ethanolic extracts of Cardiospermum halicacabum L. leaf

S. No. Concentrations (μg/ml) Superoxide radical Scavenging activity (%)
Standard (Ascorbic acid) Chloroform extract Ethanol extract
1. 100 12.18±0.85 10.00±0.7 6.25±0.43
2. 200 19.22±1.34 16.66±1.16 15.62±1.09
3. 300 35.32±2.47 33.33±2.33 18.75±1.31
4. 400 55.34±3.87 53.33±3.73 40.00±2.8
5. 500 72.24±5.05 70.00±4.90 56.25±3.93

IC50 value of chloroform extract = 380 µg/ml, IC50 value of ethanol extract = 460 µg/ml


Table 6: ABTS radical scavenging activity of chloroform and ethanolic extracts of Cardiospermum halicacabum L. leaf

S. No. Concentrations (μg/ml) ABTS radical Scavenging activity (%)
Standard (Ascorbic acid) Chloroform extract Ethanol extract
1. 100 10.38±0.72 7.14±0.49 6.89±0.48
2. 200 22.46±1.57 10.71±0.74 20.83±1.45
3. 300 27.46±1.92 25.00±1.75 24.14±168
4. 400 39.66±2.77 32.14±2.24 37.93±2.65
5. 500 48.28±3.37 46.42±3.24 44.82±3.13
6. 600 60.20±4.21 57.14±3.99 51.72±3.62

IC50 value of chloroform extract = 530 µg/ml, IC50 value of ethanol extract = 570 µg/ml

The ABTS cation is scavenged by the plant extract of Dimocarpus longan Lour.has significant antioxidant potential. The ABTS assay principle resembles the DPPH assay. The mode of action of both assay are similar. The antioxidant potential increases with increase in concentration of the plant extract [22]. ABTS has been used to evaluate the antioxidative activity ofnatural products in organic solvents. Hydrogen-donating abilityof the antioxidant is responsible for its free radical-scavenging activity. The ABTS method is based on the reduction of ABTS in the presence of a hydrogen-donating antioxidant. The aqueous extract of Ricinus communisL. leaves and the isolated compounds showed a concentration dependent antiradical activity. The aqueous extract of the leaves was found to possess radical-scavenging activity. Ascorbic acid was used as positivecontrol [27]. The Ehrlich tumor was initially described as a spontaneous murine mammary adenocarcinoma. It is a rapidly growing carcinoma with very aggressive behavior and is able to grow in almost all strains of mice [34]. Hence EAC cell is used in the present study to screen the anticancer potential of the selected plant drug. The chloroform and ethanol extracts of the leaves of Cardiospermum halicacabum L. was tested against EAC cell line. Different concentration of plant (chloroform and ethanol) extracts was inoculated with selected cell line and the cytotoxicity was assessed using trypan blue dye exclusive method. The test based on the principle that the dead cell accepts dye and stain with blue colour. The plant drug may disturb the membrane integrity and caused the cell death, which is one of the hall marks of apoptosis. The chloroform and ethanol extracts showed 73.71% and 60.19% of cytotoxicity (1000 µg/ml) against EAC cell line (Table 7, 8). The alcoholic extract of Premna herbacea has showed a significant antitumor activity against MCF-7 and EAC cell line was detected by using trypan blue dye method. The plant extract encounters the cancer cells by inducing the apoptosis [35]. In vitro cytotoxicity study was carried out by the chloroform and ethanolic extracts of Cardiospermum halicacabum L. against EAC cell line employing MTT assay method. The reduction of tetrazolium salts is now widely accepted as a reliable way to examine the cell proliferation. The yellow tetrazolium MTT (3- (4, 5 dimethyl thiazolium -2) - 2, 5 diphenyltetrazolium bromide) is reduced by metabolic active cells by the action of mitochondrial dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazon can be solubilized and quantified by spectrophotometric method [36].

Table 7: Cytotoxic effect of chloroform extract of Cardiospermum halicacabum L. leaf on EAC cell line (Trypan Blue Method)

S. No.CONCENTRATIONS (µg/ml)VIABLE CELLS (%)DEAD CELLS (%)
1. Control 96.15±6.73 3.84±0.26
2. 100 85.00±5.95 15.00±1.05
3. 250 76.00±5.32 24.00±1.68
4. 500 64.00±4.48 36.00±2.52
5. 750 47.31±3.31 52.69±3.68
6. 1000 26.28±1.83 73.71±5.15

Dead cell= Stained with Trypan blue dye, Viable cell= Not stained with Trypan blue dye


Table 8: Cytotoxic effect of ethanolic extract of Cardiospermum halicacabum L. leaf on EAC cell line (Trypan Blue Method)

S. No.Concentration (µg/ml)Viable cells (%)Dead cells (%)
1. Control 97.00±6.79 3.00±0.21
2. 100 85.50±5.98 14.50±1.01
3. 250 75.44±5.28 24.56±1.71
4. 500 67.00±4.69 33.00±2.31
5. 750 58.31±4.08 41.69±2.91
6. 1000 39.81±2.78 60.19±4.21

Dead cell= Stained with Trypan blue dye, Viable cell= Not stained with Trypan blue dye


Table 9: Cytotoxic effect of chloroform extract of Cardiospermum halicacabum L. leaf against EAC cell line (MTT assay)

S. No.Concentration (µg/ml)OD 1OD 2OD 3Average%
1. Control 0.377±0.02 0.414±0.028 0.304±0.021 0.365±0.025 -
2. 50 0.307±0.02 0.327±0.022 0.341±0.023 0.325±0.022 10.95±0.766
3. 75 0.283±0.01 0.276±0.019 0.259±0.018 0.272±0.019 25.47±1.78
4. 100 0.244±0.01 0.232±0.016 0.211±0.014 0.229±0.016 37.26±2.60
5. 125 0.198±0.01 0.194±0.013 0.191±0.013 0.194±0.013 46.75±3.27
6. 150 0.183±0.01 0.174±0.012 0.162±0.011 0.173±0.012 52.60±3.68
7. 200 0.125±0.008 0.136±0.009 0.125±0.008 0.127±0.008 65.11±4.55

IC50 value of chloroform extract = 140 µg/ml


Table 10: Cytotoxic effect of ethanolic extract of Cardiospermum halicacabum L. leaf against EAC cell line (MTT assay)

S. No.CONCENTRATIONS (µg/ml)OD 1OD 2OD 3AVERAGE%
1. Control 0.466±0.032 0.453±0.031 0.461±0.0322 0.460±0.032 -
2. 50 0.499±0.034 0.323±0.022 0.397±0.027 0.406±0.028 11.73±0.821
3. 75 0.370±0.025 0.366±0.025 0.348±0.024 0.361±0.025 21.52±1.50
4. 100 0.295±0.020 0.311±0.021 0.318±0.022 0.308±0.021 33.04±2.312
5. 125 0.283±0.019 0.255±0.017 0.241±0.016 0.259±0.018 43.69±3.058
6. 150 0.245±0.017 0.223±0.015 0.218±0.015 0.228±0.015 50.43±3.530
7. 200 0.191±0.013 0.185±0.012 0.184±0.012 0.186±0.013 59.56±4.169

IC50 value of ethanol extract = 150 µg/ml

From the result, it was observed that the chloroform and ethanolic extracts of plant under study has potent cytotoxicity and maximum cytotoxicity 65.11% and 59.56%was found at the concentration of 200µg/ml and the IC50 values were found to be 140µg/ml and 150µg/ml respectively (Table 9 & 10). The plant drug might have disturbs the mitochondrial assembly which resulted in the increased cytotoxicity of EAC cell line. In vitro cytotoxicity of brucine, a natural plant alkaloid was analyzed against the EAC cell line and human cancer cell line by using MTT assay method.

The brucine shows a significant cytotoxicity against the EAC cell line and human cancer cell line. The brucine may disturbs the mitochondrial membrane and leads to cancer cell death. In vitro cytotoxicity of ethanolic extractof Cordiaver benacea leaves was analyzed against the EAC cell line by using the MTT assay method. It destructs the mitochondrial membrane and induced the apoptosis of cancer cells [37]. The results of the present study concluded that the preliminary phytochemical screening of Chloroform extract contains alkaloids, coumarin, flavones, quinones, saponins, steroids and tannins. Ethanol extract showed the presence of alkaloids, coumarin, flavones, quinones, saponin, terpenoids, steroids and sugar. In vitro antioxidant studies were carried out for the chloroform and ethanol extracts of the test drug using various free radical models such a DPPH, Reducing power assay, NO, H2O2, SOD and ABTS. The results revealed that the chloroform extract has significant antioxidant potential than ethanol extract. In vitro cytotoxic assay such as trypan blue dye exclusion and MTT assays were carried out against EAC cell line. The result revealed that the chloroform extracts of Cardiospermum halicacabum L. showed pronounced anticancer activity against Ehrlich Ascites Carcinoma (EAC) cell line than ethanol extract.

CONFLICT OF INTERESTS

Declared None

ACKNOWLEDGEMENT

The authors are grateful to Dr. S. Velavan, Director, Harman Institute of Science Education and Research (www. Harman researchcentre. com), Thanjavur, Tamil Nadu for their support.

REFERENCES

  1. Siegel AB and Zhu AX. Metabolic syndrome and hepatocellular carcinoma:two growing epidemics with a potential link. J Cancer 2009;115:5651-61.
  2. Kushi LH, Doyle C and Mc Cullough M. American Cancer Society Guidelines on nutrition and physical activity for cancer prevention:reducing the risk of cancer with healthy food choices and physical activity. CA Cancer J Clin 2012;62 (1):30-67.
  3. Kinzler, Kenneth Vogelstein and Bert. The genetic basis of human cancer. J New York:Mc Graw-Hill, Medical Pub 2002;005-009.
  4. Kuper H, Boffetta P, Adami HO. Tobacco use and cancer causation:association by tumour type. J of Internal Medicine 2002;252 (3):206-24.
  5. Ellestad GA. Structural and conformational features relevant to the anti-tumor activity of caliche amiciny. J Chirality 2011;23, 660-71.
  6. Benedek B, Ziegler A, Ottersbach P. Absence of mutagenic effects of a particular Symphytum officinale L. liquid extract in the bacterial reverse mutation assay. Phytotherapy Res 2010;24:466-8.
  7. Jeyadevi R, Sivasudha T, Rameshkumar A, James Harnly and Long-Ze Lin. Phenolic profiling by UPLC–MS/MS and hepatoprotective activity of Cardiospermum halicacabum against CCl4 induced liver injury in Wistar rats. J of Functional Foods 2013;289-98.
  8. Venkatesh Babu KC, Krishnakumari S. Cardiospermum halicacabum suppresses the production of TNF-alpha and Nitric oxide by Human Peripheral Blood Mononuclear cells. African J of Biomedical Res 2006;9:95-99.
  9. Abdul Waheed, Yamin Bibi, Sobia Nisa, Fayyaz Chaudhary, Sumaira Sahreen and Muhammad Zia. Inhibition of human breast and colorectal cancer cells by Viburnum foetens L. extracts in vitro. Asian Pacific J of Tropical Disease 2013;3(1):32-6.
  10. Girish H, Sudarshana MS, Rati Rao E. In vitro Evaluation of the Efficacy of Leaf and its Callus Extracts of Cardiospermum halicacabum L. On Important Human Pathogenic Bacteria. J Advances in Biological Res 2008;2 (1-2):34-8.
  11. Brindha P, Sasikala, Bhima Rao. Pharmacognostic studies on Coleus Aromaticus Benth. J Indian Borage 1981;12:17-31.
  12. Gyamfi MA, Rati Rao E, Aniya Y. Antioxidant properties of thonningianin A, isolated from the African medicinal herb. Thonningia sanguine. J Biochem Pharmacol 2002;63:1725-37.
  13. Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Jap J Nutr 1986;44:307-15.
  14. Ruch RJ, Chung SU, Klaunig J E. Spin trapping of superoxide and hydroxyl radicals. J Methods in Enzymology 1984;105:198-209.
  15. Nathan CF, Rati Rao P, Hibbs JJB. Role of nitric oxide synthesis in macrophage antimicrobial activity. J Curr Opin Immunol 1991;3:65-70.
  16. Nishimiki M, Rao N A, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. J Biochem Biophys Res Commun 1972;46:849-53.
  17. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. J Free Radical Biology and Medicine 1999;26:231–1237.
  18. Fangli Hou, Murray C J and Ezzati M. Hepatoprotective and antioxidant activity of anthocyanins in black rice bran on carbon tetrachloride-induced liver injury in mice. J of Functional Foods 2013;1705-13.
  19. Gothoskar S V and Ranadive K J. Anticancer screening of SAN-AB, An extract of marking nut Semicarpus anacardium. Indian J Exp Biol 1971;9:372-5.
  20. Sheeja K R, Kuttan G and Kuttan R. Cytotoxic, antitumour activity of Berberin. J Amala Res. Bull 1997;17:73-6.
  21. Scudiero DA, Shoemaker RH and Paul KD. Evaluation of soluble terazolium formazan assay for cell growth and drug sensitivity in clusters using human and other tumor cell lines. J Cancer Res 1988;48:4827-33.
  22. Nagendra Prasad, Shahira Ezzat and Laila Ismail. Antioxidant and anticancer activities of high pressure-assisted extract of longan (Dimocarpus longan Lour.) fruit pericarp. J Innovative Food Sci and Emerging Technologies 2009;10:413-9.
  23. Raju Senthil Kumar, Balasubramanian Rajkapoor and Perumal Perumal. In vitro and in vivo anticancer activity of Indigo feracassioides Rottl. Ex. DC. Asian Pacific J of Tropical Medicine 2011;379-85.
  24. Rajesh Kumar G, Murugananthan K, Nandakumar K, Sahil Talwar. “Isolation of anxiolytic principle from ethanolic root extract of Cardiospermum halicacabum”. J Phytomedicine 2011;219-23.
  25. Mohammad Sekendar Ali, Mohammad Ruhul Amin, Chowdhury Mohammad Imtiaz Kamal, Mohammad Aslam Hossain. In vitro antioxidant, cytotoxic, thrombolytic activities and phytochemical evaluation of methanol extract of the A. philippense L. leaves. Asian Pacific J of Tropical Biomedicine 2013;3(6):464-9.
  26. Nesrine Ouda Amari, Mohamed Bouzouina, Abdellah Berkani, Brahim Lotmani. Phytochemical screening and antioxidant capacity of the aerial parts of Thymelaea hirsute L. Asian Pacific J of Tropical Disease 2014;4(2):104-9.
  27. Pradeep Pratap Singh, Ambika and Chauhan S M S. Activity guided isolation of antioxidants from the leaves of Ricinus communis L. J Food Chemistry 2009;1069-72.
  28. Velavan Sivanandham. Free Radicals In Health And Diseases-A Mini Review. J Pharmacologyonline Newsletter 2011;1062-77.
  29. Nabila Benariba, Murray CJ. and Ezzati M. Phytochemical screening and free radical scavenging activity of Citrullus colocynthis seeds extracts. Asian Pacific J of Tropical Biomedicine 2013;3(1):35-40.
  30. Shahidi F and Zhong Y. Novel antioxidants in food quality preservation and health Promotion. European J of Lipid Sci and Technology 2010;112, 930-40.
  31. Rabia Naz and Asghari Bano. Phytochemical screening, antioxidants and antimicrobial potential of Lantana camara in different solvents. Asian Pacific J of Tropical Disease 2013;3(6):480-6.
  32. Miek Jonga, Ulrike Ermuth and Matthias Augustin. Plant-based ointments versus usual care inthe management of chronic skin diseases:a comparative analysis on outcome and safety. J Complementary Therapies in Medicine 2013;453-9.
  33. Hussein Farhan, Murray CJ, Ezzati M. “Phytochemical screening and antioxidant activity of Lebanese Eryngium creticum L. Asian Pacific J of Tropical Biomedicine 2012;S1217-S20.
  34. Constantin T, Konstantinos Dimas, Zacharias Sofianos. Metabolism and Anticancer Activity of the Curcumin Analogue, Dimethoxycurcumin. J Clinical Cancer Res 2007;13:1269-77.
  35. Isha Dhamija, Nitesh Kumar, Manjula S N., Vipan Parihar, Manjunath Setty M, Pai R. Preliminary evaluation of in vitro cytotoxicity and in vivo antitumor activity of Premna herbacea Roxb. in Ehrlich ascites carcinoma model and Dalton’s lymphoma ascites model. J Experimental and Toxicologic Pathology 2013;235-42.
  36. Ursula Sauer, Shahira Ezzat, Laila Ismail. Applicability of rat precision-cut lung slices in evaluating nanomaterial cytotoxicity, apoptosis, oxidative stress, and inflammation. J Toxicology and Applied Pharmacology 2014;1–20.
  37. Eduardo Parisotto, Murray CJ, Ezzati M. The antitumor activity of extracts from Cordia verbenacea D.C. obtained by supercritical fluid extraction. The J of Supercritical Fluids 2012;101-7.